Polo-like kinases (Plks) are important regulators of mitotic progression, and abnormal Plk function has been implicated in the pathogenesis of different tumor types. In the budding yeast, Saccharomyces cerevisiae, Cdc5 is the only Plk, and is essential for mitotic exit. Despite the importance of Plks in mitosis, the regulation of kinase activity remains poorly understood. The research proposed here will address this problem by exploiting the ability to purify both active and inactive forms of Cdc5 in budding yeast. First, proteins that specifically associate with active Cdc5 will be identified by mass spectrometry, and tested as regulators of Cdc5 activity in kinase assays and genetic experiments. Second, the major sites of phosphorylation on active Cdc5 will be mapped by mass spectrometry. Identification of these sites will enable a series of Cdc5 phosphosite mutants to be generated, which will be tested for kinase activity in vitro, and for the ability to rescue the late mitotic arrest of the cdc5-1 strain. Finally, inactive Cdc5 will be used as a substrate to purify Cdc5-activating kinase(s) from extracts of wild-type yeast by standard chromatographic techniques. These studies will permit the identification and characterization of physiologically relevant regulatory proteins that control the activity of Cdc5.